Compositions and methods for selective inhibition of nicotine acetylcholine receptors

ABSTRACT

The present invention concerns methods for treating or preventing neurological disorders characterized by dysfunction of nicotinic acetylcholine receptors by administering 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), or a pharmaceutically acceptable salt thereof, to the patient. In another aspect, the present invention pertains to pharmaceutical compositions containing TMPH, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In another aspect, the present invention pertains to methods for selectively inhibiting nicotinic acetylcholine receptors that lack an α5, α6, or β3 subunit by contacting an effective amount of TMPH, or a pharmaceutically acceptable salt thereof, to the receptor. The method for selectively inhibiting nicotinic acethylcholine receptors that lack an α5 subunit can be carried out in vivo or in vitro.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application Ser. No.60/507,744, filed Oct. 1, 2003, which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, and drawings.

The subject invention was made with government support under a researchproject supported by National Institutes of Health Grant NS32888 andNational Institutes of Mental Health Grant MH11258. Accordingly, thegovernment has certain rights in this invention.

BACKGROUND OF INVENTION

There are multiple types of nicotine acetylcholine receptors (nAChR) inthe brain associated with synaptic function, signal processing, or cellsurvival. The therapeutic targeting of nicotinic receptors in the brainwill benefit from the identification of drugs which may be selective fortheir ability to activate or inhibit a limited range of these receptorsubtypes. Mecamylamine is a ganglionic blocker developed many years agoas an antihypertensive and more recently suggested to be useful as acomponent in the pharmacotherapy for Tourette's syndrome (Sanberg, P. R.et al., Lancet, 1998, 352:705-706) and smoking cessation (Rose, J. E. etal., Clin. Pharmacol. Ther., 1994, 56(1):86-99). However,electrophysiological characterization of mecamylamine has shown it to berelatively nonselective (Papke, R. L. et al., J Pharmacol Exp Ther,2001, 297(2):646-56), consistent with the observation that iteffectively blocks all of the peripheral and central nervous system(CNS) effects of nicotine (Martin, B. R. et al., Med. Chem. Res., 1993,2:564-577).

A family of bis-tetramethylpiperidine compounds have been identified asinhibitors of neuronal-type nicotinic receptors (Francis, M. M. et al.,Biophys. J., 1998, 74(5):2306-2317). The prototype compound in thisseries is bis-(2,2,6,6-tetramethyl-4-piperidinyl)-sebacate (BTMPS orTinuvin 770), which produces a readily reversible block of muscle-typenAChR and a nearly irreversible use-dependent, voltage-independent blockof neuronal nAChR. The tetramethyl-piperidine groups of BTMPS aresufficient to inhibit nAChR, and the conjugation of two such groups by along aliphatic chain accounts for both the selectivity and slowreversibility of BTMPS inhibition of neuronal nAChR (Francis, M. M. etal., 1998).

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns methods of treating a patient sufferingfrom a neurological condition, such as Tourette's syndrome or other ticdisorders, characterized by the dysfunction of nicotine acetylcholinereceptors (nAChRs), by administering 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (also referred to herein as TMPH or compound I), or apharmaceutically acceptable salt thereof, to the patient. The use ofTMPH facilitates the development of therapies for a number ofneurological disorders, with improved selectivity for nAChR subtypes.

According to the method of the present invention, TMPH can beadministered as an isolated compound, or administered in apharmaceutically acceptable carrier as a pharmaceutical composition ofthe subject invention. Optionally, TMPH can be administered with otherpharmacologically active agents, such as nicotinic acetylcholinereceptor agonists, antagonists, or mixed agonists/antagonists.

In other aspects, the present invention concerns a compound comprisingTMPH or a pharmaceutically acceptable salt thereof; and pharmaceuticalcompositions containing TMPH, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.

In another aspect, the present invention pertains to methods forselectively inhibiting nicotinic acetylcholine receptors that lack an α5subunit, an α6 subunit, or β3 subunit by contacting an effective amountof TMPH, or a pharmaceutically acceptable salt thereof, to the receptor.For example, neuronal beta subunit-containing receptors without an α5subunit, α6 subunit, or β3 subunit can be inhibited. Receptors such asmuscle-type (α1β1γδ) and α7 receptors, can be inhibited. The method canbe carried out in vivo or in vitro. For example, an effective amount ofTMPH, or a pharmaceutically acceptable salt thereof, can be administeredin vivo to a patient in need thereof, in order to treat a neurologicalcondition, such as nicotine addiction. Alternatively, an effectiveamount of TMPH, or a pharmaceutically acceptable salt thereof, can becontacted in vitro to isolated nicotine acetylcholine receptors thatlack an α5, α6, or β3 subunit or to cells that naturally orrecombinantly express nicotine acetylcholine receptors that lack an α5,α6, or β3 subunit.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show the dose-response blockade of nicotine-inducedantinociception in the tail-flick (FIG. 1A) and the hot-plate test (FIG.1B) by TMPH after s.c. injection in mice. TMPH at different doses wasadministered s.c. 10 min before nicotine (2.5 mg/kg, s.c.) and mice weretested 5 min later. Each point represents the mean±SE of 8 to 12 mice.

FIG. 2 shows the time-course of TMPH effect on nicotine-inducedantinociception (2.5 mg/kg) in (

) the tail-flick and (

) the hot-plate tests after s.c. administration of 5 mg/kg in mice. Eachpoint represents the mean±SE of 8 to 12 mice. *p<0.05 compared tocorrespondent zero time point.

FIGS. 3A-3B show the effects of PC-7 in combination with 0.4 mg/kgnicotine on percentage of nicotine-lever responding (FIG. 3A) andresponse rates (FIG. 3B) in rats trained to discriminate 0.4 mg/kgnicotine from vehicle. Points above V and N represent the results ofcontrol tests with two saline injections and saline plus 0.4 mg/kgnicotine, respectively, conducted before the dose-effect curvedetermination. Each value represents the mean (+SEM) of 4-6 rats.

FIGS. 4A-4B show that TMPH produces selective long-term inhibition ofneuronal ganglionic type α3β4 receptors. In FIG. 4A, the upper panelshows raw data traces obtained from an oocyte expressing muscle-typeα1β1εδ subunits to the application of either 10 μM ACh alone (opencircles) or the co-application of 10 μM ACh and 300 nM TMPH (arrow). Thelower panel is the averaged normalized data (±SEM, n≧4) from oocytesexpressing α1β1εδ subunits to the co-application 10 μM ACh and a rangeof TMPH concentrations. In FIG. 4B, the upper panel shows raw datatraces obtained from an oocyte expressing ganglionic-type α3β4 subunitsto the application of either 100 μM ACh alone (open circles) or theco-application of 100 μM ACh and 300 nM TMPH (arrow). The lower panel isthe averaged normalized data (±SEM, n≧4) from oocytes expressing α3β4subunits to the co-application 100 μM ACh and a range of TMPHconcentrations. Three values are plotted in each of theconcentration-response curves: (♦) the peak current amplitude of theco-application response, normalized to the peak amplitude of theprevious ACh control; (▾) the net charge of the co-application response,normalized to the net charge of the previous ACh control (Papke andPapke, 2002); and (●) the peak current amplitude of the ACh controlresponse obtained after the TMPH/ACh co-application, normalized to thepeak amplitude of the previous ACh control.

FIGS. 5A-5C show that TMPH produces long-term inhibition of neuronalbeta subunit-containing nAChR but not α7 homomeric receptors. Shown arethe averaged normalized data (±SEM, n≧4) from oocytes expressing α4β2,α3 β2, and α7 subunits (FIGS. 5A-5C, respectivley) to the co-applicationACh and a range of TMPH concentrations. Three values are plotted in eachof the concentration-response curves: (♦) the peak current amplitude ofthe co-application response, normalized to the peak amplitude of theprevious ACh control; (▾) the net charge of the co-application response,normalized to the net charge of the previous ACh control; and (●) thepeak current amplitude of the ACh control response obtained after theTMPH/ACh co-application, normalized to the peak amplitude of theprevious ACh control. The control ACh concentrations used were 10 μM, 30μM, and 300 μM for α4β2, α3β2, and α7, respectively.

FIG. 6 shows that the recovery of α7 receptors from TMPH-producedinhibition is rapid compared to the recovery of neuronal betasubunit-containing nAChR. ACh and TMPH was co-applied at time 0 tooocytes expressing rat α3β4, α4β2, α3β2, or α7 subunits. Subsequently,control ACh applications were made at 5 min intervals to measurerecovery. The control ACh concentrations used were 100 μM, 10 μM, 30 μM,and 300 μM for α3β4, α4β2, α3β2, and α7, respectively. The initialinhibition was produced by the co-application of ACh at the controlconcentration and 30 μM TMPH, except in the case of the α3β2 receptors,which were inhibited by the co-application of ACh and 3 μM TMPH.

FIGS. 7A-7B show varying amounts of use-independent inhibition ofneuronal beta subunit-containing receptors by TMPH. FIG. 7A containsrepresentative data showing the effects of the application of 1 μM TMPHalone to α4β2 and α3β2 expressing oocytes (open arrows), compared to theapplication of to ACh alone (open circles) either before or after theapplication of TMPH. The control ACh concentrations used were 10 μM and30 μM for α4β2 and α3β2, respectively. FIG. 7B shows comparison of theuse-dependent (1 μM TMPH+ACh) and use-independent (1 μM TMPH alone)inhibition of α3β4, α4β2, and α3β2 receptors by TMPH. The data arecalculated from the peak amplitudes of control ACh responses obtainedafter the application of TMPH±ACh, expressed relative to the peakcurrent amplitude of ACh control responses prior to the application ofTMPH.

FIGS. 8A-8B show cumulative inhibition by repeated application of TMPH.FIG. 8A shows responses of an oocyte expressing α4β2 receptors toalternating applications of 30 μM ACh alone (open circles) or 30 μM AChplus 100 nM TMPH (arrows). The total inhibition increased during thefirst 3 TMPH/ACh co-applications. Note that the TMPH/ACh co-applicationsdiffer in kinetics from the ACh controls, showing a greater inhibitionof net charge than of peak current. FIG. 8B shows normalized averageresponses of oocytes expressing α4β2 receptors (±SEM, n≧4) toalternating applications of ACh alone or ACh plus TMPH, as in FIG. 8A.Both peak currents and net charge values are plotted, normalized to theACh control response recorded before the first ACh/TMPH co-application(t=−5 minutes). Note that the traces in FIG. 8A are shown on the sametime scale as the X-axis in FIG. 8B.

FIGS. 9A-9B show TMPH inhibition of responses obtained from oocytesexpressing human neuronal nAChR subunits. Shown are the averagednormalized data (±SEM, n≧4) from oocytes expressing human α3β2 or α3β2α5subunits (top and bottom, respectively) to the co-application ACh and arange of TMPH concentrations. Three values are plotted in each of theconcentration-response curves: (♦) the peak current amplitude of theco-application response, normalized to the peak amplitude of theprevious ACh control; (▾) the net charge of the co-application response,normalized to the net charge of the previous ACh control; and (●) thepeak current amplitude of the ACh control response obtained after theTMPH/ACh co-application, normalized to the peak amplitude of theprevious ACh control. The control ACh concentrations used were 30 μM and1 μM for α3β2 and α3β2α5 expressing oocytes, respectively.

FIGS. 10A-10B show the recovery of human α5-containing α3β2 receptorsfrom TMPH-produced inhibition is rapid compared to the recovery ofreceptors formed with α3β2 subunits alone. FIG. 10A shows representativetraces obtained from oocytes expressing human α3β2 or α3β2α5 subunits.Following an initial application of ACh alone (open circle), a singleco-application was made of ACh and 1 μM TMPH (arrow). Recovery wasevaluated by making repeated control ACh applications at 5 minuteintervals (open circles). FIG. 10B shows normalized average responses ofoocytes expressing α3β2 or α3β2α5 receptors (±SEM, n≧4) to repeatedapplications of ACh alone, following a single application of ACh plusTMPH (as in FIG. 10A). Peak currents are plotted, normalized to the AChcontrol response recorded before the ACh/TMPH co-application. Note thatthe traces in FIG. 10A are shown on the same time scale as the X-axis inFIG. 10B.

FIGS. 11A-11C show TMPH inhibition of responses obtained from oocytesexpressing β3 and chimeric α6/α3 subunits. Shown are the averagednormalized data (±SEM, n≧4) from oocytes expressing human α3β2β3 orα6/3β2β3 subunits (FIGS. 11A and 11B, respectively) to theco-application ACh and a range of TMPH concentrations. Three values areplotted in each of the concentration-response curves: (♦) the peakcurrent amplitude of the co-application response, normalized to the peakamplitude of the previous ACh control; (▾) the net charge of theco-application response, normalized to the net charge of the previousACh control; and (●) the peak current amplitude of the ACh controlresponse obtained after the TMPH/ACh co-application, normalized to thepeak amplitude of the previous ACh control. The control AChconcentration used was 100 μM.

FIGS. 12A-12B show TMPH inhibition of responses obtained from oocytesexpressing b4, β3 and either α3 (FIG. 12A) or α6 subunits (FIG. 12B).Shown are the averaged normalized data (±SEM, n≧4) to the co-application100 μM ACh and a range of TMPH concentrations. Three values are plottedin each of the concentration-response curves: (♦) the peak currentamplitude of the co-application response, normalized to the peakamplitude of the previous ACh control; (▾) the net charge of theco-application response, normalized to the net charge of the previousACh control; and (●) the peak current amplitude of the ACh controlresponse obtained after the TMPH/ACh co-application, normalized to thepeak amplitude of the previous ACh control. Note that the recovery datafor oocytes expressing α3, β4, and β3 could not be fit to a one sitemodel. The curve fit show is for a 2 site model with approximately 15%fit to an IC₅₀ of 1 μM and 85% fit to an IC₅₀ of 30 μM.

DETAILED DISCLOSURE OF THE INVENTION

The present inventors have determined that2,2,6,6-tetramethylpiperidin-4-yl heptanoate (also referred to herein asTMPH or compound I), a compound that has a single tetramethyl-piperidinegroup and an aliphatic chain, is a potent inhibitor of neuronalnicotinic receptors. Moreover, when delivered systemically, TMPH canblock the effects of nicotine on the central nervous system (CNS),indicating that this drug is able to cross the blood-brain barrier andaccess sites in the brain. Surprisingly, however, unlike the prototypeCNS-active nicotinic inhibitor, mecamylamine, TMPH blocks only some ofthe CNS effects of nicotine, indicating that it has a unique selectivityfor specific receptor subtypes in the brain. Since non-selectivenicotinic inhibitors with CNS activity have been suggested to bepotentially useful treatments for neuropsychiatric disorders and forpromoting smoking cessation, a more selective agent, such as TMPH, canprovide therapeutic approaches with a reduced range of side effects.Additionally, based on the characterization of this agent's effects onspecific nAChR subtypes, TMPH may identify the particular molecularsubstrates that underlie the multiple effects of nicotine on the brain.TMPH (compound I) is shown below.

The subject invention concerns methods of treating a patient sufferingfrom a neurological condition characterized by the dysfunction ofnicotine acetylcholine receptors (nAChRs) by administering2,2,6,6-tetramethylpiperidin-4-yl heptanoate (also referred to herein asTMPH or compound I), or a pharmaceutically acceptable salt thereof, tothe patient.

The fact that nAChRs having an α5, α6, or β3 subunit are spared from theinhibitory effects of TMPH permits the tuning of the selectivity ofspecific compounds to increase desired effects and diminish sideeffects. For example, the selective inhibition of nAChRs lacking an α5,α6, or β3 subunit by administration of TMPH, or a pharmaceuticallyacceptable salt thereof, permits the avoidance of side effects (e.g.,increased heart rate and increased blood pressure) normally associatedwith the inhibition of nAChRs containing these subunits by non-selectiveinhibitors, such as Tinuvin 770 and mecamylamine. As used herein, theterms α5, α6, and β3 subunits include the human nicotinic acetylcholinereceptor subunits and mammalian homologs of the same name (Chini et al.,Proc. Natl. Acad. Sci. USA 89:1572-1576).

Preferably, the compounds (TMPH, or a pharmaceutically acceptable saltthereof) and compositions of the subject invention are administered totreat a patient suffering from a neurological disorder associated withdysfunction of one or more subtypes of nAChR, or to prevent onset of thedisorder. Neurological disorders which can be treated or prevented withpharmaceutical compositions of the present invention, and in accordancewith methods of the present invention, include, but are not limited to,Tourette's syndrome or other tic disorders, presenile dementia (earlyonset Alzheimer's disease), senile dementia (dementia of the Alzheimer'stype), Parkinsonism including Parkinson's disease, Huntington's chorea,tardive dyskinesia, hyperkinesias, mania, attention deficit disorder,attention deficit hyperactivity disorder, sleep-wake disorder,chronic-fatigue syndrome, tremor, epilepsy, neuropathic pain, addiction(e.g., nicotine/smoking addiction), anxiety, dyslexia, schizophrenia,and obsessive-compulsive disorder (Salamone, F. et al., MJM, 2000,5:90-97; Cooper, E. C. and Jan, L. Y. Proc. Natl. Acad. Sci. USA, 1999,96:4759-4766; Sharples, C. and Wonnacott, S. Neuronal NicotinicReceptors, October 2001, 19:1-12; Mihailescu, S. and Drucker-Colin, R.Arch. Med. Res., 2000, 31:131-144; Papke, R. L. et al., Euro. J. Pharm.,2000, 393:179-195; Newhouse, P. A. and Kelton, M. Pharm. Acta Helv.,2000, 74:91-101; Newhouse, P. A. et al., Clin. Pharm., 1997, 11:206-228;Lloyd, G. K. and Williams, M. J. Pharm. Exp. Therapies, 2000,292:461-467; Hollady, M. W. et al., J. Med. Chem., 1997, 40:4169-4194;Benowitz, N. L. Annu. Rev. Pharm. Toxicol., 1996, 36:597-613; Freedman,R. et al., Harvard Rev. Psych., 1994, 2:179-192; and Freedman, R. etal., J. Chem. Neuroanatomy, 2000, 20:299-306).

In a specific embodiment, the method of the present invention involvesadministration of TMPH, or a pharmaceutically acceptable salt thereof,to a patient as adjunctive therapy in order to treat or prevent Tourettesyndrome (TS) or other tic disorder, such as transient or chronic ticdisorders. As described in A Physician's Guide to Diagnosis andTreatment of Tourette Syndrome (published by the Tourette SyndromeAssociation, Inc., 1984), TS is characterized by multiform, frequentlychanging motor and phonic tics. The prevailing diagnostic criteriainclude onset before the age of 21; recurrent, involuntary, rapid,purposeless motor movements affecting multiple muscle groups; one ormore vocal tics; variations in the intensity of the symptoms over weeksto months (waxing and waning); and a duration of more than one year. Ofcourse, these criteria are not absolute. The varied symptoms of TS canbe divided into motor, vocal, and behavioral manifestations. Motorsymptoms can include simple motor tics, which are fast, darting, andmeaningless, and complex motor tics, which are slower, may appearpurposeful (includes copropraxia and echopraxia). Vocal symptoms caninclude simple vocal tics, which are meaningless sounds and noises, andcomplex vocal tics, which are linguistically meaningful utterances, suchas words and phrases (including coprolalia, echolalia, and palilalia).Behavior and developmental symptoms can include attention deficithyperactivity disorder, obsessions and compulsions, emotional lability,irritability, impulsivity, aggressivity, self-injurious behaviors, andvaried learning disabilities. The methods and compositions of thepresent invention can be used to prevent or lessen the severity of oneor more of these symptoms. Preferably, the methods and compositions ofthe present invention are used as an adjunctive therapy in combinationwith other pharmacologic treatments for TS, such as haloperidol(HALDOL), pimozide (ORAP) or other neuroleptics, clonidine (CATAPRESE),clomipramine, fluoxetine (PROZAC), and combinations thereof. Forexample, compositions containing TMPH, or a pharmaceutically acceptablesalt thereof, and one or more of the aforementioned drugs areencompassed by the invention and may be administered in accordance withthe methods of the invention.

In another embodiment of the method of the present invention, TMPH or apharmaceutically acceptable salt thereof, is administered to a patientfor treatment or prevention of nicotine addition. TMPH or apharmaceutically acceptable salt thereof can be administered withanother pharmacologically active compound to treat or prevent nicotineaddiction. For example, TMPH or a pharmaceutically acceptable saltthereof can be administered to a patient in combination with nicotine(e.g., nicotine can be transdermally administered by application of anicotine patch).

Mammalian species which benefit from the disclosed methods of treatmentinclude, and are not limited to, apes, chimpanzees, orangutans, humans,monkeys; domesticated animals (e.g., pets) such as dogs, cats, guineapigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. The term“patient” is intended to include such human and non-human mammalianspecies.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Furthermore, as used herein, the phrase“pharmaceutically acceptable carrier” means any of the standardpharmaceutically acceptable carriers. The pharmaceutically acceptablecarrier can include diluents, adjuvants, and vehicles, as well asimplant carriers, and inert, non-toxic solid or liquid fillers,diluents, or encapsulating material that does not react with the activeingredients of the invention. Examples include, but are not limited to,phosphate buffered saline, physiological saline, water, and emulsions,such as oil/water emulsions. The carrier can be a solvent or dispersingmedium containing, for example, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils. Formulations containingpharmaceutically acceptable carriers are described in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Sciences (Martin E W,Remington's Pharmaceutical Sciences, Easton Pa., Mack PublishingCompany, 19^(th) ed., 1995) describes formulations that can be used inconnection with the subject invention. Formulations suitable forparenteral administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The TMPH or pharmaceutically acceptable salt thereof can be produced bymethods known in the art for synthesis of hindered amine compounds.

TMPH, or a pharmaceutically acceptable salt thereof (or a pharmaceuticalcompositions containing TMPH or a pharmaceutically acceptable saltthereof), can be administered to a patient by any route that results inprevention or alleviation of symptoms associated with the particularneurological condition. For example, as described in more detail below,TMPH or a pharmaceutically acceptable salt thereof can be administeredparenterally, intravenously (I.V.), intramuscularly (I.M.),subcutaneously (S.C.), intradermally (I.D.), orally, intranasally, etc.Examples of intranasal administration can be by means of a spray, drops,powder or gel. However, other means of drug administrations are wellwithin the scope of the present invention.

The pharmaceutical compositions disclosed herein may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or they may be compressed into tablets, or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compounds may be incorporated with excipientsand used in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, TMPH or apharmaceutically acceptable salt thereof may be incorporated intosustained-release preparation and formulations.

TMPH may also be administered parenterally or intraperitoneally.Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases of injection, the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenyl, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating TMPH, or apharmaceutically acceptable salt thereof, in the required amount in theappropriate solvent with other various ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Examples of “pharmaceutically acceptable carriers” include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. In one embodiment, the pharmaceutically acceptable carrieris a sterile, fluid (e.g., liquid or gas) preparation rendering thepharmaceutical composition suitable for injection or inhalation. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

For oral prophylaxis, TMPH or a pharmaceutically acceptable saltthereof, may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, TMPH or a pharmaceutically acceptable saltthereof, may be incorporated into an antiseptic wash containing sodiumborate, glycerin and potassium bicarbonate. TMPH or a pharmaceuticallyacceptable salt thereof may also be dispersed in dentifrices, including:gels, pastes, powders and slurries. TMPH or a pharmaceuticallyacceptable salt thereof may be added in a therapeutically effectiveamount to a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains an active ingredient is well understood in theart. Typically, such compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified.

The composition of the present invention can be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

According to the therapeutic methods of the present invention, TMPH, ora pharmaceutically acceptable salt thereof, is administered (broughtinto contact with nAChRs lacking α5, α6, or β3 subunits in vivo) anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight, and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. Fortherapeutic methods, the amount of TMPH, or a pharmaceuticallyacceptable salt thereof, must be effective to achieve improvementincluding but not limited to total prevention and to improved survivalrate or more rapid recovery, or improvement or elimination of symptomsassociated with the particular neurological condition, such asTourette's syndrome or other tic disorders, and other indicators as areselected as appropriate measures by those skilled in the art. Inaccordance with the present invention, a suitable single dose size is adose that is capable of preventing or alleviating (reducing oreliminating) a symptom in a patient when administered one or more timesover a suitable time period. One of skill in the art can readilydetermine appropriate single dose sizes for systemic administrationbased on the size of a mammal and the route of administration.Optionally, the therapeutic methods of the invention further comprisediagnosis of the patient with a neurological condition by a medicalpractitioner (e.g., a medical doctor, veterinarian, or other clinician).The therapeutic methods may further comprise evaluating the patient forone or more symptoms associated with the neurological condition beforeand/or after administration of TMPH or a pharmaceutically acceptablesalt thereof.

The methods and compositions of the invention may incorporate additionalpharmacologically active agents (such as for adjunctive therapy), inaddition to TMPH or a pharmaceutically acceptable salt thereof. Forexample, the additional pharmacologically active agent can beco-administered consecutively or simultaneously (e.g., in the sameformulation or different formulations.

In one embodiment, the additional pharmacologically active agent is annAChR modulator, such as an inhibitor of nAChR activity. Preferably, theagent is a selective inhibitor. In one embodiment, the agent is aTMPH-related compound, its structure differing from TMPH in having asingle tetramethyl piperidine group and an aliphatic chain that iseither longer or shorter than that of TMPH (e.g., having a hydrocarbonlength of 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15 . . . etc.).

As used herein, the term “treatment” or grammatical variations thereofis intended to mean reducing (e.g., lessening or eliminating) orpreventing of one or more symptoms associated with a particularneurological disorder characterized by dysfunction (e.g., overactivationor overexpression) of one or more subtypes of nAChR.

As used herein, “activity” of a nAChR refers to any activitycharacteristic of a nAChR. Such activity can typically be measured byone or more in vitro methods, and frequently corresponds to an in vivoactivity of a nAChR. Thus, the terms “function” and “activity” withrespect to nicotine AChR means that the receptor channel is able toprovide for and regulate entry of nicotinic AChR-permeable ions, suchas, for example, Na⁺, K⁺, Ca²⁺, or Ba²⁺, in response to a stimulusand/or bind ligands with affinity for the receptor. Preferably, suchnicotinic AChR activity is distinguishable, such as byelectrophysiological, pharmacological, and other means known to those ofskill in the art, from the endogenous nicotinic AChR activity that maybe produced by the host cell in the absence of TMPH or apharmaceutically acceptable salt thereof. As used herein, the term“inhibit” with respect to nAChR activity is intended to include partialor complete inhibition of nAChR activity.

In another aspect, the present invention pertains to methods forselectively inhibiting nicotinic acetylcholine receptors that lack anα5, α6, or β3 subunit by contacting an effective amount of TMPH, or apharmaceutically acceptable salt thereof, to the receptor or combinationof receptor subunits in vitro or in vivo. For example, neuronal betasubunit-containing receptors lacking an α5, α6, or β3 subunit can beinhibited. Receptors such as muscle-type (α1β1γδ), and α7 receptors, canbe inhibited. The method can be carried out in vivo or in vitro. Forexample, an effective amount of TMPH, or a pharmaceutically acceptablesalt thereof, can be administered in vivo to a patient in need thereof.Alternatively, an effective amount of TMPH, or a pharmaceuticallyacceptable salt thereof, can be contacted in vitro to nicotineacetylcholine receptors that lack an α5, α6, or β3 subunit or to cellsthat naturally or recombinantly express nicotine acetylcholine receptorsthat lack an α5, α6, or β3 subunit. Optionally, the method furthercomprises determining nAChR activity before, during, or after the TMPHor a pharmaceutically acceptable salt thereof is contacted with thenAChR subunit(s). The effect of TMPH or a pharmaceutically acceptablesalt thereof on a particular nAChR subunit combination can be determinedby comparison of the change in receptor function (e.g., byelectrophysiological recordings). The nAChR subunits can be mammalian,such as human. Examples of host cells appropriate for recombinantexpression of neuronal nAChR subunits include, but are not limited to,bacterial cells (e.g., Escherichia coli), yeast cells (e.g.,methylotrophic yeast cells, such as Pichia pastoris), and mammaliancells (e.g., HEK 293, CHO and Ltk⁻ cells). The cells to which TMPH or apharmaceutically acceptable salt thereof may be contacted in vitroinclude isolated cells, cell cultures, cell lines, tissues (e.g., tissuecultures), etc.

In other preferred embodiments, eukaryotic cells which containheterologous nucleic acid sequences encoding nicotinic AChR(s) expressthe heterologous nucleic acid sequences and form recombinant functionalnicotinic AChR(s). In more preferred aspects, recombinant nicotinic AChRactivity is readily detectable because it is a type that is absent fromthe untransfected host cell or is of a magnitude not exhibited in theuntransfected cell. Such cells that contain recombinant receptors can beprepared, for example, by causing cells transformed with DNA encodingnicotinic AChR α₃ and β₄ subunits to express the corresponding proteins.The resulting synthetic or recombinant receptor would contain only theα₃ and β₄ nAChR subunits. Testing of single receptor subunits with TMPH,or a pharmaceutically acceptable salt thereof, can provide additionalinformation with respect to the function and activity of the individualsubunits in response to this selective inhibitor. Such information maylead to the identification of other compounds which are capable of veryspecific interaction with one or more of the receptor subunits. Suchspecificity may prove of great value in medical application.

Thus, nucleic acid sequences encoding one or more nicotinic AChRsubunits (e.g., human nicotinic AChR subunits) may be introduced intosuitable host cells (e.g., eukaryotic or prokaryotic cells) forexpression of individual subunits and functional AChRs. Preferably,combinations of alpha and beta subunits may be introduced into cells.Such combinations include any combinations of any one or more of α₁, α₂,α₃, α₄, and α₇, with β₂ and/or β₄. Sequence information for α₁ ispresented in Biochem. Soc. Trans. (1989) 17:219-220; sequenceinformation for α₅ is presented in Proc. Natl. Acad. Sci. USA (1992)89:1572-1576; and sequence information for α₂, α₃, α₄, α₇, β₂ and β₄ ispresented elsewhere in the literature. Preferred combinations ofsubunits include any one or more of α₁, α₂, or α₃, with β₄; or α₄ or α₇in combination with either β₂ or β₄. It is recognized that some of thesubunits may have ion transport function in the absence of additionalsubunits. For example, the α₇ subunit is functional in the absence ofany added beta subunit.

As used herein, the term “expression” refers to the process by whichnucleic acid sequences are transcribed into mRNA and translated intopeptides, polypeptides, or proteins. If the polynucleic acid is derivedfrom genomic DNA, expression may, if an appropriate eukaryotic host cellor organism is selected, include splicing of the mRNA. Particularlypreferred vectors for transfection of mammalian cells are the pSV2dhfrexpression vectors, which contain the SV40 early promoter, mouse dhfrgene, SV40 polyadenylation and splice sites and sequences necessary formaintaining the vector in bacteria, cytomegalovirus (CMV) promoter-basedvectors such as pCDNA1 (INVITROGEN, San Diego, Calif.), and MMTVpromoter-based vectors such as pMSG (Catalog No. 27-4506-01 fromPHARMACIA, Piscataway, N.J.).

Nicotinic AChR subunits and cells producing them may be tested by themethods provided herein or known to those of skill in the art to detectfunctional AChR activity. Such testing will allow the identification ofpairs of alpha and beta subunit subtypes that produce functional AChRs,as well as individual subunits that produce functional AChRs. As usedherein, activity of a nAChR refers to any activity characteristic of anAChR. Such activity can typically be measured by one or more in vitromethods, and frequently corresponds to an in vivo activity of a nAChR.Such activity may be measured by any method known to those of skill inthe art, such as, for example, measuring the amount of current whichflows through the recombinant channel response to a stimulus. Methods todetermine the presence and/or activity of nicotinic AChRs include assaysthat measure nicotine binding, ⁸⁶Rb ion-flux, Ca²⁺ influx, theelectrophysiological response of cells, the electrophysiologicalresponse of oocytes transfected with RNA from the cells, and the like.

Modulation of neuronal nicotinic AChR activity in response to contactwith TMPH or a pharmaceutically acceptable salt thereof can involvecomparison to a control. One type of a “control” cell or “control”culture is a cell or culture that is treated substantially the same asthe cell or culture exposed to the test compound, except the controlcell or culture is not exposed to the test compound (TMPH or apharmaceutically acceptable salt thereof). For example, in methods thatuse voltage clamp electrophysiological procedures, the same cell can betested in the presence and absence of TMPH or a pharmaceuticallyacceptable salt thereof, by merely changing the external solutionbathing the cell. Another type of “control” cell or “control” culturemay be a cell or a culture of cells which are identical to thetransfected cells, except the cells employed for the control culture donot express functional human neuronal nicotinic AChRs. In thissituation, the response of the test cell to the TMPH or pharmaceuticallyacceptable salt thereof is compared to the response (or lack ofresponse) of receptor-negative (control) cell to the TMPH orpharmaceutically acceptable salt, when cells or cultures of each type ofcell are exposed to substantially the same reaction conditions in thepresence of compound being assayed.

In one embodiment, amphibian oocytes (e.g., Xenopus oocytes) are usedfor expression of one or more nAChR subunits, and an effective amount ofTMPH, or a pharmaceutically acceptable salt thereof, is contacted withthe oocytes. Methods for injecting oocytes and performingelectrophysiological and other analyses for assessing receptorexpression and function are described herein. While methods for in vitrotranscription of cloned DNA and injection of the resulting mRNA intoeukaryotic cells are well known in the art, amphibian oocytes areparticularly preferred for expression of in vitro transcripts of nAChRDNA clones, including human nAChR DNA clones. See, for example, Dascal(CRC Crit. Rev. Biochem., 1989, 22:317-387), for a review of the use ofXenopus oocytes to study ion channels.

Throughout the subject application, nicotinic acetylcholine receptorsubunits are referred to by a numeral preceded by the letter of theGreek alphabet or an abbreviation. For example, the α5 subunit may alsobe referred to as “alpha5” or “a5”.

Throughout the subject application, TMPH may be substituted with achemical analog in connection with the compound, composition, andmethods of the present invention. As used herein, the term “analogs”refers to compounds which are substantially the same as another compoundbut which may have been modified by, for example, adding side groups,oxidation or reduction of the parent structure. Analogs of theexemplified compounds can be readily prepared using commonly knownstandard reactions. These standard reactions include, but are notlimited to, hydrogenation, alkylation, acetylation, and acidificationreactions.

As used in this specification, including the appended claims, thesingular “a”, “an”, and “the” include plural reference unless thecontact dictates otherwise. Thus, for example, a reference to “asubunit” includes more than one such subunit. A reference to “a cell”includes more than one such cell. A reference to “a receptor” includesmore than one such receptor, and so forth.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term.

As used herein, the term “additional pharmacologically active agent”refers to any agent, such as a drug, capable of having a physiologiceffect (e.g., a therapeutic or prophylactic effect) on prokaryotic oreukaryotic cells, in vivo or in vitro, including, but withoutlimitation, chemotherapeutics, toxins, radiotherapeutics,radiosensitizing agents, gene therapy vectors, antisense nucleic acidconstructs or small interfering RNA, imaging agents, diagnostic agents,agents known to interact with an intracellular protein, polypeptides,and polynucleotides.

The additional pharmacologically active agent can be selected from avariety of known classes of drugs, including, for example, analgesics,anesthetics, anti-inflammatory agents, anthelmintics, anti-arrhythmicagents, antiasthma agents, antibiotics (including penicillins),anticancer agents (including Taxol), anticoagulants, antidepressants,antidiabetic agents, antiepileptics, antihistamines, antitussives,antihypertensive agents, antimuscarinic agents, antimycobacterialagents, antineoplastic agents, antioxidant agents, antipyretics,immunosuppressants, immunostimulants, antithyroid agents, antiviralagents, anxiolytic sedatives (hypnotics and neuroleptics), astringents,bacteriostatic agents, beta-adrenoceptor blocking agents, blood productsand substitutes, bronchodilators, buffering agents, cardiac inotropicagents, chemotherapeutics, contrast media, corticosteroids, coughsuppressants (expectorants and mucolytics), diagnostic agents,diagnostic imaging agents, diuretics, dopaminergics (antiparkinsonianagents), free radical scavenging agents, growth factors, haemostatics,immunological agents, lipid regulating agents, muscle relaxants,proteins, peptides and polypeptides, parasympathomimetics, parathyroidcalcitonin and biphosphonates, prostaglandins, radio-pharmaceuticals,hormones, sex hormones (including steroids), time release binders,anti-allergic agents, stimulants and anoretics, steroids,sympathomimetics, thyroid agents, vaccines, vasodilators, and xanthines.

The additional pharmacologically active agent need not be a therapeuticagent. For example, the agent may be cytotoxic to the local cells towhich it is delivered but have an overall beneficial effect on thesubject. Further, the agent may be a diagnostic agent with no directtherapeutic activity per se, such as a contrast agent for bioimaging.

MATERIALS AND METHODS

Synthesis. Chemicals used for the synthesis were purchased from ALDRICHChemical Company. Compounds were characterized by ¹H-NMR and FAB-MS.

TMPH synthesis. To a mixture of 2,2,6,6-tetramethyl-4-piperidinol (472mg, 3.0 mmol) and methyl heptanoate (476 mg, 3.3 mmol) in 3.0 mL ofdimethyl formamide was added 250 mg of powdered potassium carbonate. Theresulting mixture was heated at 145˜155° C. for 64 hours under a gentlestream of N₂. After cooling, the reaction mixture was partitionedbetween water and hexanes. The organic layer was separated, washed withwater (2×) and brine, then dried over anhydrous MgSO₄ and evaporated toafford the crude product as an oil. The oil was dissolved in MeOH andwas then treated with 2 equivalents of conc. HCl. The solvent wasremoved in vacuo, and the residue was then treated with diethyl ether.The resulting solids were removed by filtration. The ethereal filtratewas concentrated in vacuo and triturated with hexane to afford 380 mg(41%) of TMPH hydrochloride. It was recrystallized from boiling ethylacetate/hexane to afford short colorless needles, mp 113-115° C.FAB-HRMS: calculated (C₁₆H₃₂NO₂): 270.2433 found: 270.2435.

In Vivo Studies

Animals. Male ICR mice (20-25 g) obtained from Harlan Laboratories(Indianapolis, Ind.) were used throughout the study. Animals were housedin groups of six and had free access to food and water. Adult, maleLong-Evans rats (350-460 g), obtained from Harlan (Dublin, Va.), wereindividually housed in a temperature-controlled (20-22° C.) environmentwith a 12-hour light-dark cycle (lights on at 7 a.m.). Rats weremaintained within the indicated weight range by restricted post-sessionfeeding and had ad libitum water in their home cages. Rats weredrug-naive at the beginning of the study. Animals were housed in anAALAC approved facility and the study was approved by the InstitutionalAnimal Care and Use Committee of Virginia Commonwealth University.

Drugs. Mecamylamine hydrochloride was supplied as a gift from Merck,Sharp and Dohme & Co. (West Point, Pa.). (−)-Nicotine was obtained fromAldrich Chemical Company, Inc. (Milwaukee, Wis.) and converted to theditartrate salt as described by Aceto et al. (Aceto et al. J Med Chem,1979, 22:174-177. All drugs were dissolved in physiological saline (0.9%sodium chloride). All doses are expressed as the free base of the drug.

Behavioral Assays

Locomotor activity. Mice were placed into individual Omnitech photocellactivity cages (28×16.5 cm) immediately after s.c. administration ofeither 0.9% saline or nicotine (6.2 μmol/kg or 1 mg/kg) and were allowedto acclimate for 10 minutes. Interruptions of the photocell beams (twobanks of eight cells each) were then recorded for the next 10 minutes.Data were expressed as percentage of depression where %depression=[1−(counts from nicotine-treated animals/counts fromvehicle-treated animals)]×100. Mice were pretreated s.c. with eithersaline or TMPH 10 minutes before nicotine.

Antinociception.

1. Tail-flick test. Antinociception was assessed by the tail-flickmethod of D'Amour and Smith (D'Amour and Smith J. P. E. T., 1941,72:74-79) as modified by Dewey et al. (Dewey et al. J Pharmacol ExpTher, 1970, 175:435-442). Briefly, mice were lightly restrained while aradiant heat source was shone onto the upper portion of the tail.Latency to remove the tail from the heat source was recorded for eachanimal. A control response (2-4 seconds) was determined for each mousebefore treatment, and a test latency was determined after drugadministration. In order to minimize tissue damage, a maximum latency of10 seconds was imposed. Antinociceptive response was calculated aspercent maximum possible effect (% MPE), where %MPE=[(test-control)/(10-control)]×100.

2. Hot-plate Test. Mice were placed into a 10 cm wide glass cylinder ona hot plate (Thermojust Apparatus) maintained at 55.0° C. Two controllatencies at least 10 minutes apart were determined for each mouse. Thenormal latency (reaction time) was 8 to 12 seconds. Antinociceptiveresponse was calculated as percent maximum possible effect (% MPE),where % MPE=[(test-control)/(40-control)×100]. The reaction time wasscored when the animal jumped or licked its paws. In order to minimizetissue damage, a maximum latency of 40 seconds was imposed. Antagonismstudies were carried out by pretreating the mice with either saline orTMPH 10 minutes before nicotine. The animals were tested 5 minutes afteradministration of nicotine.

Body temperature. Rectal temperature was determined by a thermistorprobe (inserted 24 mm) and digital thermometer (Yellow SpringsInstrument Co., Yellow Springs, Ohio). Readings were taken just beforeand 30 minutes after the s.c. injection of nicotine at a dose of 12.3μmol/kg (2 mg/kg). Mice were pretreated with either saline or TMPH(s.c.) 10 minutes before nicotine. The difference in rectal temperaturebefore and after treatment was calculated for each mouse. The ambienttemperature of the laboratory varied from 21-24° C. from day to day.

The doses of nicotine used in the different tests representapproximately an ED₈₄ (Effective dose 84%) which were determined fromprevious works (Damaj et al. Psychopharmacology (Berl), 1995,117:67-73). Eight to twelve mice were tested in each treatment group andeach animal was tested only once.

Drug Discrimination in Rats.

1. Apparatus: Rats were trained and tested in standard operantconditioning chambers (Lafayette Instruments Co., Lafayette, Ind.)housed in sound-attenuated cubicles. Each chamber had three retractablelevers, only two of which were used for this study. Pellet dispensersdelivered 45-mg BIO SERV (Frenchtown, N.J.) food pellets to a food cupon the front wall of the chamber between the two response levers andover the third (retracted) lever. Fan motors provided ventilation andmasking noise for each chamber. House lights located above the food cupwere illuminated during training and testing sessions. A micro-computerwith Logic ‘1’ interface (MED Associates, Georgia, Vt.) and MED-PCsoftware (MED Associates) was used to control schedule contingencies andto record data.

2. Procedure: Rats were trained to press one lever followingadministration of 0.4 mg/kg nicotine and to press another lever afterinjection with saline, each according to a fixed-ratio 10 schedule offood reinforcement. Completion of 10 consecutive responses on theinjection-appropriate lever resulted in delivery of a food reinforcer.Each response on the incorrect lever reset the ratio requirement on thecorrect lever. The position of the drug lever was varied among the groupof rats. The daily injections for each rat were administered in a doublealternation sequence of 0.4 mg/kg nicotine and saline. Rats wereinjected and returned to their home cages until the start of theexperimental session 5 minutes later. Training occurred during sessionsconducted five days a week (Monday-Friday) until the rats had met threecriteria during eight of ten consecutive sessions: (1) first completedfixed ratio 10 on the correct lever, (2) percentage of correct-leverresponding >80% for the entire session, and (3) response rate >0.4responses/sec.

Following successful acquisition of the discrimination, stimulussubstitution tests with test compounds were conducted on Tuesdays andFridays during 15-minute test sessions. Training continued on Mondays,Wednesdays, and Thursdays. During test sessions, responses on eitherlever delivered reinforcement according to a fixed ratio 10 schedule. Inorder to be tested, rats must have completed the first FR and made atleast 80% of all responses on the injection-appropriate lever on thepreceding day's training session. In addition, the rat must have metthese same criteria during at least one of the training sessions withthe alternate training compound (nicotine or saline) earlier in theweek.

A nicotine dose-effect determination [0.1, 0.2, 0.4, 0.8, and 1.2 mg/kg]was performed first in each rat. Then, combination tests with nicotine,and TMPH followed (see figures for specific doses). Doses of eachcompound were administered in ascending order. Throughout the study,control tests with saline and 0.4 mg/kg nicotine were conducted duringthe week before the start of each dose-effect curve determination.

Statistical analysis. Statistical analysis of all analgesic and in vivostudies was performed using either t-test or analysis of variance(ANOVA) with Tukey's test post hoc test when appropriate. Alldifferences were considered significant at p<0.05. AD₅₀ values with 95%CL for behavioral data were calculated by unweighted least-squareslinear regression as described by Tallarida and Murray (Tallarida andMurray Manual of Pharmacological Calculations with Computer Programs.1987, Springer-Verlag, New York).

Expression in Xenopus oocates

Animals: Xenopus

Mature (>9 cm) female Xenopus laevis African frogs (NASCO, Ft. Atkinson,Wis.) were used as a source of oocytes. Prior to surgery, frogs wereanesthetized by placing the animal in a 1.5 g/l solution of MS222(3-aminobenzoic acid ethyl ester) for 30 minutes. Oocytes were removedfrom an incision made in the abdomen.

In order to remove the follicular cell layer, harvested oocytes weretreated with 1.25 mg/ml collagenase from Worthington BiochemicalCorporation (Freehold, N.J.) for 2 hours at room temperature incalcium-free Barth's solution (88 mM NaCl, 10 mM HEPES pH 7.6, 0.33 mMMgSO₄, 0.1 mg/ml gentamicin sulfate). Subsequently, stage 5 oocytes wereisolated and injected with 50 nl (5-20 ng) each of the appropriatesubunit cRNAs. Recordings were made 2 to 15 days after injection.

Preparation of RNA

Rat neuronal nAChR clones and mouse muscle nAChR cDNA clones were used.The wild-type clones were obtained from Dr. Jim Boulter (UCLA). The ratα6/3 (Dowell et al. J Neurosci, 2003, 23:8445-8452) clone was obtainedfrom Michael McIntosh (University of Utah) and expressed in Xenopusoocytes in combinations with rat β2 and β3. The original α6/3 constructprovided was sequenced and was found to have a mutation in the secondtransmembrane domain (TM2) sequence which exchanged a valine for analanine in the 7′ position (TM2 numbering scheme (Miller, Neuron, 1989,2:1195-1205)). The TM2 domain is understood to line the pore of thechannel, with alpha helix structure. The 7′ position may actually bedirected away from the actual pore lining, but this residue is highlyconserved in all of the nAChR. It is valine in all of them except α9(isoleucine) and β1, where it is alanine. The TM2 mutation in the α6/3chimera was corrected by using QuickChange (Stratagene) according totheir protocols. The corrected α6/3 chimera sequence was confirmed byrestriction diagnostics and by automated fluorescent sequencing(University of Florida core facility). The corrected clone was expressedas above in Xenopus oocytes with β2 and β3 and compared to the wild-typeα3 co-expressed with β2 and β3.

After linearization and purification of cloned cDNAs, RNA transcriptswere prepared in vitro using the appropriate mMessage mMachine kit fromAmbion Inc. (Austin, Tex.).

Electrophysiology. The majority of experiments were conducted usingOpusXpress 6000A (Axon Instruments, Union City Calif.). OpusXpress is anintegrated system that provides automated impalement and voltage clampof up to eight oocytes in parallel. Cells were automatically perfusedwith bath solution, and agonist solutions were delivered from a 96-wellplate. Both the voltage and current electrodes were filled with 3 M KCl.The agonist solutions were applied via disposable tips, which eliminatedany possibility of cross-contamination. Drug applications alternatedbetween ACh controls and experimental applications. Flow rates were setat 2 ml/min for experiments with α7 receptors and 4 ml/min for othersubtypes. Cells were voltage-clamped at a holding potential of −60 mV.Data were collected at 50 Hz and filtered at 20 Hz. Agonist applicationswere 12 seconds in duration followed by 181-second washout periods forα7 receptors and 8 seconds with 241-second wash periods for othersubtypes. For some experiments, particularly under conditions whereresidual inhibition precluded making repeated measurements from singlecells (see below), manual oocyte recordings were made as previouslydescribed (Papke and Papke Br J of Pharmacol, 2002, 137:49-61). Inbrief, Warner Instruments (Hamden, Conn.) OC-725C oocyte amplifiers wereused, and data were acquired with a minidigi or digidata 1200A withpClamp9 software (Axon Instruments). Sampling rates were between 10 and20 Hz and the data were filtered at 6 Hz. Cells were voltage clamped ata holding potential of −50 mV. Data obtained with these methods werecomparable to those obtained with OpusXpress.

Experimental protocols and data analysis. Each oocyte received twoinitial control applications of ACh, an experimental drug application(or co-application of ACh and TMPH), and then follow-up controlapplication(s) of ACh. The control ACh concentrations for α1β1γδ, α3β4,α4β2, α3β2, α3β2α5, α3β2β3, α6/3β2β3 α6β4β3 and α7, receptors were 30μM, 100 μM, 10 μM, 30 μM, 1 μM 100 μM 100 μM 100 μM and 300 μM,respectively. In other experiments (Papke et al. J. Neurochem., 2000,75:204-216; Papke and Papke Br J of Pharmacol, 2002, 137:49-61) theseconcentrations were determined to be the EC₇₄, EC₁₅, EC₂₂, EC₁₇, EC₄₀,EC₇₀, EC₅₅, EC₇₀ and EC₁₀₀, respectively. These concentrations wereselected since they gave large responses with relatively littledesensitization so that the same oocyte could be stimulated repeatedlywith little decline in the amplitude of the ACh responses. This allowedthe inhibitory effects of the antagonist to be separated out frompossible cumulative desensitization.

Responses to experimental drug applications were calculated relative tothe preceding ACh control responses in order to normalize the data,compensating for the varying levels of channel expression among theoocytes. Responses were characterized based on both their peakamplitudes and the net charge (Papke and Papke Br J of Pharmacol, 2002,137:49-61). In brief, for net charge measurement a 90-second segment ofdata beginning 2 seconds prior to drug application was analyzed fromeach response. Data were first adjusted to account for any baselineoffset by subtracting the average value of 5-second period of baselineprior to drug application from all succeeding data points. Whennecessary, baseline reference was also corrected for drift usingClampfit 9.0 (AXON Instruments, Union City Calif.). Following baselinecorrection, net charge was then calculated by taking the sum of all theadjusted points. The normalized net charge values were calculated bydividing the net charge value of the experimental response by the netcharge value calculated for the preceding ACh control response. Meansand standard errors (SEM) were calculated from the normalized responsesof at least 4 oocytes for each experimental concentration. In order tomeasure the residual inhibitory effects, this subsequent controlresponse was compared to the pre-application control ACh response.

For concentration-response relations, data derived from net chargeanalyses were plotted using Kaleidagraph 3.0.2 (Abelbeck Software;Reading, Pa.), and curves were generated from the Hill equation

$\text{Response} = \frac{{I_{\max}\lbrack{agonist}\rbrack}^{n}}{\lbrack{agonist}\rbrack^{n} + \left( {{EC}50} \right)^{n}}$where I_(max) denotes the maximal response for a particularagonist/subunit combination, and n represents the Hill coefficient.I_(max), n, and the EC₅₀ were all unconstrained for the fittingprocedures. Negative Hill slopes were applied for the calculation ofIC₅₀ values.

EXAMPLE 1 Effects of TMPH on Nicotine's Actions In Vivo

Antinociception

Nicotine-induced antinociception in the tail-flick and hot-plate testsafter systemic administration in mice (2.5 mg/kg) was blocked by TMPH ina dose-dependent manner (FIGS. 1A-1B). Calculation of the AD₅₀ showedthat TMPH is 1.7 times more potent in blocking the antinociceptiveeffect of nicotine in the hot-plate than in tail-flick test (0.7 versus1.2 mg/kg). By itself, TMPH after s.c. injection did not causeantinociception at the indicated doses and times.

Time-Course of TMPH Effects

The duration of action of TMPH in the tail-flick test was time-dependentwith maximum blockade occurring between 15 and 30 minutes after a doseof 5 mg/kg dose. The effect of TMPH lasted for at least 4 hours afterits administration. Indeed, as illustrated in FIG. 2, nicotine's effectstarted to recover within 60 minutes after pretreatment with a dose of 5mg/kg of TMPH, but was still significantly different from control 4hours after. Similar to the tail-flick test, TMPH time-dependentlyblocked nicotine-induced antinociception as measured by the hot-platetest, however with a shorter duration of action. As shown in FIG. 2, 60minutes after pretreatment with a dose of 5 mg/kg of TMPH the effect ofnicotine recovered fully to the pre-treatment value.

Locomotor Activity and Body Temperature

TMPH at 20 mg/kg administered s.c. 15 minutes prior to the injection ofnicotine (1.5 mg/kg) failed to significantly reduce the hypomotilityinduced by nicotine (Table 1). In addition, nicotine-induced hypothermiaafter systemic administration in mice (2.5 mg/kg) was also not blockedby TMPH given at 20 mg/kg. By itself, TMPH after s.c. injection did nothave a significant effect on the body temperature or the locomotoractivity at the indicated doses and times.

TABLE 1 Effect of TMPH on nicotine-induced hypomotility and hypothermiaafter s.c. administration. Each point represents the mean ± SE of 6 to 8mice. Locomotor Activity Body temperature Treatment # Interrupts Δ° C.(mg/kg) (Mean ± SEM) (Mean ± SEM) Saline/Saline 1931 ± 120 −0.3 ± 0.1TMPH (20)/Saline 2031 ± 160 −1.0 ± 0.2 Saline/Nicotine (1.5)  358 ± 92*−5.0 ± 0.3* TMPH (20)/Nicotine (1.5)  397 ± 170* −5.2 ± 0.4* *P < 0.05from Saline/SalineNicotine Discriminative Stimulus in Rats

FIGS. 3A-3B shows the results of combination tests with the trainingdose of nicotine and various doses of TMPH. TMPH dose-dependentlyantagonized the discriminative stimulus effects of 0.4 mg/kg nicotine(FIG. 3A) with an AD₅₀ value of 0.74 mg/kg (0.56-0.98) (Table 2). TheTMPH-nicotine combination did not alter response rates (compared tovehicle) at any of the dose combinations tested (p>0.05; FIG. 3B). TMPHalone also did not produce nicotine-lever responding at the doses atwhich antagonism was observed (data not shown).

TABLE 2 Comparison of the blockade potency of TMPH and mecamylamine onnicotine's pharmacological and behavior effects after systemic andadministration in mice and rats. TMPH^(a) Mecamylamine^(b) Test (AD₅₀mg/kg ± CL) (AD₅₀ mg/kg ± CL) Tail-flick  1.2 (0.6-1.8) 0.045 (0.03-0.1)Hot-plate  0.7 (0.3-1.7)  0.8 (0.5-1.1) Drug discrimination 0.74(0.56-0.98)  0.91 (0.63-1.32) Hypothermia 0% blockade @ 20  1.2(0.9-1.8) Hypomotility 0% blockade @ 20  1.95 (1.1-2.5) ^(a)AD₅₀ values(±CL) were calculated from the dose-response and expressed as mg/kg.Each dose group included 6 to 8 animals. ^(b)AD₅₀ values (±CL) weretaken from Damaj et al. (Psychopharmacology (Berl), 1995, 117: 67-73)and Wiley et al. (Exp Clin Psychopharmacol, 2002, 10: 129-135).

EXAMPLE 2 Effects of TMPH on nAChR Expressed in Xenopus Oocytes

TMPH Inhibition of AChR Subtypes

TMPH was initially tested on mouse muscle-type (α1β1εδ) nAChR, threedifferent pairwise combinations of rat neuronal alpha and beta subunits(α3β4, α4β2, and α3β2), and α7 homomeric neuronal nAChR. Additionallycombinations of three subunits and an α6/3 chimera were tested. Theresults are summarized in Table 3. As shown in FIGS. 4A-4B, bothmuscle-type and ganglionic-type (α3β4) receptors were inhibited duringthe co-application of ACh and TMPH. However, while the inhibition ofmuscle-type receptors was readily reversible after a 5-minute wash, theinhibition of α3β4 receptors persisted after the wash. The data alsoindicate that the inhibition of α3β4 receptors became progressivelygreater during the co-application response so that the inhibition of netcharge was greater than the inhibition of peak current. This was not thecase for the inhibition of muscle-type receptors. The other neuronalalpha-beta subunit pairs tested, α4β2 and α3β2, were blocked in afashion similar to α3β4 receptors (FIGS. 5A-5C), with a largerinhibition of net charge than peak current and virtually no recoveryfollowing a 5-minute wash. In contrast, α7 receptors, like muscle-typereceptors, showed little difference between the inhibition of peakcurrents and net charge and showed significant recovery after a 5-minutewash (FIGS. 5A-5C). These differences are reflected in the IC₅₀ valuespresented in Table 3. Note that receptors that rapidly equilibrateinhibition and recover readily (e.g. muscle-type receptors and α7) haveratios of the IC₅₀ for net charge to the IC₅₀ for peak currents of closeto 1 (Table 4), while for the receptors which show progressively moreinhibition during the co-application and have slow recovery, the ratioof the IC₅₀ for net charge to the IC₅₀ for peak currents is much lessthan 1 and IC₅₀ values estimated from the inhibition of net charge aresimilar to those which can be derived from persistent inhibitionmeasured after a 5-minute wash (i.e. from the recovery data, see Table4).

TABLE 3 In vitro data. IC₅₀ (peak) IC₅₀ (area) IC₅₀ (recovery) MouseMuscle 276 ± 20 nM 390 ± 50 nM 27 ± 5.5 μM Rat α7 1.0 ± 0.1 μM 1.2 ± 0.2μM 16 ± 4 μM Rat α4β2 1.4 ± 0.3 μM 110 ± 40 nM 250 ± 80 nM Rat α3β4 200± 50 nM 75 ± 23 nM 85 ± 32 nM Rat α3β2 3.7 ± 1.2 μM 440 ± 90 nM 230 ± 30nM Human α3β2 3.7 ± 1.2 μM 460 ± 170 nM 400 ± 120 nM Human α3β2α5 1.1 ±μM 430 ± 180 nM 750 ± 200 nM Rat α3β2β3 120 ± 80 μM 2.7 ± 0.6 μM 4.3 ±1.5 μM Rat α6/3β2β3 60 ± 20 μM 1.0 ± 0.3 μM 2.3 ± 0.5 μM Rat α3β4β3 1.9± 0.2 μM 1.4 ± 0.2 μM 30 ± 50 μM, 1.0 ± 0.6 μM* Rat α6β4β3 11.0 ± 4.4 μM18.3 ± 4.9 μM 1700 ± 420 μM IC50 values were calculated based on eitherthe decrease in peak current amplitudes or the decrease in the netcharge of the ACh response when co-applied with TMPH. For many of thesubunit combinations tested there was no detectable recovery after a5-minute wash and so IC₅₀ values were also calculated based on theinhibition still present at the 5-minute time point (recovery). *Thedata for the recovery of cells expressing a3b4b3 was fit to a 2 sitemodel (see FIGS. 12A-12B).

TABLE 4 In vitro data. IC₅₀ net charge/IC₅₀ peak IC₅₀ recovery/IC₅₀ netcharge mouse α1β1δε 1.4 69 rat α7 1.2 13.3 rat α4β2 0.08 2.3 rat α3β40.36 1.1 rat α3β2 0.12 0.52 human α3β2 0.12 0.87 human α3β2α5 0.15 1.7rat α3β2β3 0.025 1.6 rat α6/3β2β3 .017 2.0 rat α3β4β3 0.74 0.7, 21* ratα6β4β3 1.7 93 The value “IC₅₀ (recovery)” reflects the residualinhibition of the ACh control response measured after a 5-minute wash.If IC₅₀ (recovery) > IC₅₀ (area), then there was significant recovery(i.e. readily reversible inhibition). Likewise if IC₅₀ (area) < IC₅₀(peak), then there was significant buildup of inhibition throughout theagonist/antagonist co-application. *The data for the recovery of cellsexpressing a3b4b3 was fit to a 2-site model (see FIGS. 12A-12B).Neuronal nAChR Recovery Rates

Initial experiments evaluated recovery after only a single 5-minutewash. In order to evaluate the actual rates at which the various nAChRsubunit combinations recovered from TMPH-induced inhibition, repeatedapplications of ACh alone after a single co-application of ACh and TMPHwere made. As shown in FIG. 6, the rat α4β2, α3β2, α3β4 showed virtuallyno detectable recovery over a period of 30 minutes, while α7 receptorswere fully recovered after about 15 minutes of wash.

Use-Dependence of Inhibition by TMPH

The degree to which inhibition by TMPH was use-dependent was determinedby applying 1 μM TMPH alone and comparing the response to a subsequentcontrol ACh application to that obtained after 1 μM TMPH was co-appliedwith ACh. As shown in FIGS. 7A-7B, the ability of TMPH to inhibitneuronal nAChR when applied in the absence of agonist variedsignificantly among the pairwise subunit combinations tested, but in allcases was less than when TMPH was co-applied with agonist.Interestingly, while TMPH alone applied to α4β2 receptors was almost aseffective as when co-applied with ACh, TMPH alone applied to α3β2receptors had no detectable effect after the washout period.

Progressive Inhibition of α4β2 Receptors by Repeated Co-Applications ofACh and TMPH Below its IC₅₀ Value

The IC₅₀ values presented in Table 3 were based on the inhibitionproduced by single co-applications (20 seconds in duration) of ACh andTMPH. Since for the neuronal beta subunit-containing receptors the onsetof inhibition is apparently much faster than the reversibility ofinhibition, measurements based on single applications of TMPH are likelyto underestimate what equilibrium IC₅₀s would be. In order to test thehypothesis that repeated applications of TMPH would produce anaccumulated inhibition that would be greater than the inhibitionproduced by a single application, repeated co-applications of ACh and100 nM TMPH to oocytes expressing α4β2 receptors were carried out.Co-applications of TMPH and ACh were alternated with applications of AChalone. As shown in FIGS. 8A-8B, repeated co-applications of ACh with 100nM TMPH (the IC₅₀ in single-dose experiments) produced 90% inhibitionafter 3 applications at 10-minute intervals. Further applications didnot produce additional inhibition. Making a corresponding shift in theα4β2 net charge inhibition curve in FIGS. 5A-5C (i.e. so that 100 nM isthe IC₉₀ rather than the IC₅₀) suggests that the equilibrium IC₅₀ wouldbe approximately 10 nM.

The Effect of α5 Co-Expression with α3β2 Subunits on the Sensitivity toTMPH

As noted above, efforts to connect data obtained from oocyte studieswith in vivo data can be complicated by the fact that in vivo nAChR mayhave more complex subunit composition than the simple pairwisealpha/beta subunit combinations most readily tested in oocytes. Anothersuch subunit that contributes to the complexity of AChRs in vivo is α5,which is not required to co-assemble with other subunits in order forthem to function but is likely to be present in some receptor subtypesin vivo (Wang et al. J. Biol. Chem., 1996, 271:17656-17665; Gerzanich etal. J. Pharmacol. Exp. Ther., 1998, 286:311-320). The hypothesis thatthe presence of the α5 subunit could modulate the sensitivity of aneuronal nAChR subunit to TMPH was tested. For these experiments, humanα3 and β2 subunits were used, which readily form receptors with orwithout the co-expression of the human α5 subunit. These subunits wereselected since the successful inclusion of the α5 subunit produces aneasily detectable change in receptor pharmacology, increasing thepotency of ACh (Gerzanich et al. J. Pharmacol. Exp. Ther., 1998,286:311-320). All batches of oocytes used for these experiments wereconfirmed to have this predicted effect of α5 expression.

As shown in FIGS. 9A-9B, ACh responses of oocytes expressing human α3β2and human α3β2α5 showed similar sensitivity to TMPH during the initialco-application. (The IC50 values based on net charge analysis were460±170 nM and 430±180 nM, respectively.) However, as shown in FIGS.10A-10B, oocytes expressing α5 along with α3 and β2 showed much fasterrecovery than those expressing α3 and β2 alone. The responses of oocytesexpressing α3β2α5 had a half time of recovery of about 15 minutes, whilesimilar to the oocytes expressing rat α3β2 receptors, those expressinghuman α3β2 receptors showed no significant recovery over a period of 40minutes.

Inhibition of Receptors Containing β3 and α6 Subunits.

It has been suggested that in vivo β3 subunits may co-assemble with α6and possibly α4 and β2 to make receptors that regulate dopamine release(Champtiaux et al. J Neurosci, 2003, 23:7820-7829). However, the α6subunit expresses poorly in oocytes when used in pairwise combinationswith beta subunits (Kuryatov et al. Neuropharmacology, 2000,39:2570-2590). Therefore, in initial experiments, in order to evaluatewhether the selective sparing of some of nicotine's effects in vivo whenTMPH is used as a blocker might be associated with receptors thatcontain α6 and/or β3, the effects of TMPH on oocytes expressing α3β2β3and a chimera of α6 and α3 subunits, α6/3 (Dowell et al. J Neurosci,2003, 23:8445-8452) were compared along with β2 and β3. This approachallows systematic evaluation of first the effects of the β3 subunits (bycomparing oocytes injected with α3β2β3 to those expressing α3β2 alone),and evaluation of the effects of the α6 extracellular domain (bycomparing oocytes injected with the α3/6 chimera in addition to β2 andβ3 to those expressing wild-type α3 along with β2 and 3β).

The addition of the β3 subunit along with α3 and β2, had the effect ofdecreasing sensitivity to an initial application of TMPH (FIG. 9A), suchthat the IC50 for inhibiting α3β2β3 receptors were at least an order ofmagnitude high than for the inhibition of α3β2 without β3 (FIGS. 5A-5Cand 11A-11C, see also Table 3). The receptors containing the α6/3chimera in combination with β2 and β3 were not significantly differentin their sensitive to TMPH than those expressing α3β2β3 wild-typesubunits (FIG. 9B and Table 3). The recovery of β3-containing receptorsfrom TMPH was relatively complex. There was about 50% recovery in thefirst 10 minutes, but no further recovery after that. This was similarfor both α3β2β3 and α6/3β2β3 (FIG. 11C). One possible explanation forthis would be if the co-expression of these subunits resulted in mixedpopulations of receptors, some containing β3 subunits, and showing rapidrecovery, and others formed without β3 and showing the nearlyirreversible block seen when α3 and β2 are expressed as a pair. This iscertainly a likely scenario for combination containing the wild-type α3,and may also be the case for the combination containing the chimera,since in the present inventors' experience there is about a two-foldincrease in currents when β3 is co-expressed with β2 and the α6/3chimera, compared to α6/3 and β2 alone (data not shown).

The data in FIGS. 11A-11C suggest that the β3 subunit imparts someresistance to inhibition by TMPH and also that the extracellular domainof α6 had relatively little effect. Therefore, oocytes expressing thecomplete wild-type α6 subunits were tested. The α6 subunit wasco-expressed with β4 and β3 since this is the only α6 combination foundto work with any consistency. For these experiments, as a control, theoocytes injected with α6β4β3 were compared to oocytes injected withα3β4β3. As shown in FIGS. 12A-12B, the oocytes injected α6β4β3 showedrelatively weak inhibition by TMPH during the co-application of TMPH andinhibitor, with an IC₅₀ for the inhibition of net charge nearly an orderof magnitude higher than for any other receptor subunit combinationtested (Table 3). This reduced sensitivity to TMPH was most likely dueto both the α6 and the β3 subunits since α3β4β3 injected oocytes weremuch less sensitive than those expressing α3β4 alone.

The responses of oocytes expressing α6β4β3 showed essentially fullrecovery after only a single wash period (FIG. 12B). However, as withthe oocytes expressing α3, β2, and β3, it is likely that the cellsinjected with α3, β4, and β3 had a mixed population of receptor sincethe recovery data was best fit with a two site model (FIG. 12A).

One of the main objectives of this study was to investigate theantagonistic effect of TMPH on central behavioral effects of nicotine,since this antagonist had not been investigated previously for itsblocking effects in vivo. Its antagonistic effects were tested on fourdifferent nicotinic responses: antinociception, discriminative cue,locomotor activity and body temperature. It has been shown that only thefirst two responses are antagonized by TMPH in a dose-related manner.The failure of TMPH to block nicotine-induced motor decrease andhypothermia suggests that it inhibits neuronal nicotinic receptors in aselective manner.

Since the systemic administration of TMPH can inhibit selective effectsof nicotine in the CNS, TMPH can apparently pass the blood-brainbarrier. The selectivity of TMPH effects in vivo suggests that it mayinhibit the effects of nicotine at some nAChR subtypes but not others,and that the nAChR subtypes which mediate the locomotor effects andhypothermic effects of nicotine are less sensitive to TMPH than thosewhich mediate analgetic effects. Based on this study of nAChR expressedin vitro, it is likely that the in vivo effects of TMPH may be due toselective inhibition of neuronal beta subunit-containing receptors thatlack the accessory subunits α5, β3, and especially α6 subunit; since theinhibition receptors containing these subunits is relatively week andreversible. Alternatively, α7-type receptors may be some of the TMPHresistant effects.

While with single applications TMPH appears to be more effective for theinhibition of α3β4 receptors than for α4β2 or α3β2 receptors, this maybe due to the relative P_(open) values during the co-applicationresponses. That is, for a use-dependent inhibitor, the fractionalinhibition increases both as more channels are open and as singlechannels multiple times (i.e. burst). If, due to their prolongedbursting behavior (Papke and Heinemann J. Physiol. (Lond.), 1991,440:95-112), proportionately more α3β4 receptors open or reopen thanα3β2 receptors during the co-application response, then the α3β4receptors will be more likely to be blocked with a given TMPHconcentration. Although the sensitivity of α3β4 receptors to TMPH mightsuggest a high liability for peripheral side effects, this may not bethe case since α5 is likely to be present in ganglionic receptors(Vernallis et al. Neuron, 1993, 10:451-464).

Compared to mecamylamine, TMPH was equipotent in blocking the effects ofnicotine in the mouse hot-plate test and the rat drug discrimination. Incontrast, mecamylamine was much more potent in blocking the othereffects of nicotine. Nicotine-induced antinociception in the hot-platetest and the nicotine discriminative stimulus were recently reported tobe largely mediated by α4β2* subtypes, that is, receptors containing α4,β2 and possibly other subunits such as α6 and β3 (Shoaib et al.Neuropharmacology, 2002, 42:530-539; Marubio et al. Eur J Neurosci,2003, 17:1329-1337). The similar potency of TMPH and mecamylamine inblocking these two nicotinic behaviors correlates well with their closepotency in blocking expressed α4β2 subtypes (TMPH and mecamylamine IC₅₀(peak) values are 1.4 and 2.5 μM, respectively). The effects on thetail-flick test seem to involve both α4β2* and non-α4β2* receptorsubtypes (Marubio et al. Eur J Neurosci, 2003, 17:1329-1337). Indeed,contrary to the hot-plate test where a nearly complete loss of theeffect was observed, nicotine-induced antinociception in the tail-flicktest showed a significant rightward shift in α4 or β2 knock-out mice(Marubio et al. Eur J Neurosci, 2003, 17:1329-1337). Compared tomecamylamine, the effects of TMPH on nicotine-induced antinociception inthe tail-flick test suggest a lower blockade potency of TMPH on thenon-α4β2* receptor subtypes. At this point, it is difficult to predictwhich nicotinic receptor subtypes are involved in this non-α4β2*component, but it seems that mecamylamine possesses much higher affinitythan TMPH to these subtypes. Recent results (Rao et al.Neuropharmacology, 1996, 35:393-405; Damaj et al. J Pharmacol Exp Ther,1998, 284:1058-1065), however, indicate little involvement of α7subtypes in the antinociceptive effects of nicotinic agonists in thetail-flick test.

The lack of TMPH effect on nicotine-induced hypomotility and hypothermiais very interesting and further indicates the in vivo selectivity ofTMPH for blocking different nicotinic receptors. The depressing effectof nicotine on locomotor activity in mice involves α5 (Salas et al. MolPharmacol, 2003, 63:1059-1066) and β2 subunits (Tritto et al. NicotineTob Res, 2004, 6:145-158), but not α4 (Salas et al. Mol Pharmacol, 2003,63:1059-1066), α7 (Tritto et al. Nicotine Tob Res, 2004, 6:145-158) andβ4 subunits as reported in recent studies using knock-out mice of thesevarious subunits. Similarly, nicotine-induced hypothermia involves β2but not α7 subunits (Tritto et al. Nicotine Tob Res, 2004, 6:145-158).The lack of effects of TMPH seems to correlate well with its relativelyweak blocking of α3α5β2 receptors. Mecamylamine and TMPH blockβ2-containing receptors with similar potency, so these results suggestthat the greater inhibitory activity of mecamylamine on nicotine-inducedantinociception may also be due to an involvement of α5-containingreceptor subtypes. Although little data are available onnicotine-induced hypothermia, the lack of TMPH's effects may possiblyinvolve similar receptor mechanisms.

In conclusion, the results suggest that drug therapies for theinhibition of CNS nicotinic receptors may be developed with greaterselectivity than previously appreciated. While more selectiveantagonists such as MLA are known, these generally work poorly withsystemic administration. Mecamylamine has previously been proposed foradjunct therapy for Tourette's syndrome (Sanberg et al. Lancet, 1998,352:705-706) and smoking cessation (Rose et al. Clin. Pharmacol. Ther.,1994, 56:86-99). The characterization of selective antagonists such asTMPH may lead the way to the development of better therapies for these,and potentially other, neuropsychiatric indications based on a morelimited profile of side effects. For example, in regard to smokingcessation, it is particularly interesting to note that TMPH blocksnicotine discrimination with a potency equal to or greater than that ofmecamylamine. However, while the concentrations of mecamylamine requiredto block drug discrimination would profoundly block potentiallydesirable antinociception mechanisms (as measured by tail-flick, Table2), concentrations of TMPH effective at blocking drug discriminationleave the effects of nicotinic receptors on tail flick responses largelyintact. In addition to the potential therapeutic significance of TMPH,this drug may also prove to be a valuable tool to combine with selectiveagonists and knockout animals to further unravel the mystery of howneuronal nicotinic receptors play a role in brain function.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method for inhibiting activity of a nicotinic acetylcholinereceptor in vivo in a patient suffering from nicotine addition, whereinsaid method comprises administering to said patient an effective amountof 2,2,6,6-tetramethylpiperidin-4-yl heptanoate, or a pharmaceuticallyacceptable salt thereof, wherein the receptor includes an alpha4 and/orbeta2 subunit; and whereby activity of the nicotinic acetylcholinereceptor is inhibited.
 2. The method of claim 1, wherein the patient isa non-human mammal.
 3. The method of claim 1, wherein the patient ishuman.
 4. The method of claim 1, wherein said administering is by aroute selected from the group consisting of intravenous, oral, andintra-nasal.
 5. The method of claim 1, wherein the2,2,6,6-tetramethylpiperidin-4-yl heptanoate or pharmaceuticallyacceptable salt thereof is administered to the patient in an amountsufficient to penetrate the blood-brain barrier.
 6. The method of claim1, wherein the nicotinic acetylcholine receptor lacks an alpha5 subunitand a beta3 subunit.
 7. The method of claim 1, wherein the nicotinicacetylcholine receptor is a neuronal beta subunit-containing receptorlacking an alpha5 subunit and a beta3 subunit.
 8. The method of claim 1,wherein the nicotinic acetylcholine receptor includes one or more of thefollowing receptor subunits: alpha3, beta4, and alpha7.
 9. The method ofclaim 1, wherein the nicotinic acetylcholine receptor includes two ormore of the following receptor subunits: alpha3, beta4, and alpha7.